There are several methods for determination of protein quantity in samples. These include use of colour-changing dyes, such as Orange G, Bromo cresol green, Pyrocatechol Violet-Molybdenum complex, etc. These dyes, when bound with protein, change colour proportional to the amount of protein present in the samples. These methods are generally not very sensitive. A more sensitive dye-binding technique using Coomassie Brilliant Blue G-200 is adversely affected by the presence of detergents in sample and also suffers from wide protein-to-protein variation (Bradford, M., Anal. Biochem., 72 248-254, 1976 and U.S. Pat. No. 4,023,933).
A variety of turbidimetric methods are also known in which protein is precipitated by various agents. These methods also suffer from lack of sensitivity, specificity and interference with detergents.
The most widely used procedures for protein determination involve the well-known reaction of protein in alkaline medium with Cupric ions yielding highly reactive cuprous ions. A method using alkaline copper was first developed by Lowry et al (Lowry, Oh. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265-275, 1951) ("the Lowry method") in which protein reaction with buffered alkaline copper was coupled with Folin phenol reagent (phosphomolybdic/phosphotungstic acid), hereinafter referred to as Folin. It is believed that protein reacts with alkaline copper and produces cuprous ions and this, in turn, reduces the Folin to the characteristic blue reaction colour.
The Lowry method suffers from many disadvantages. The most serious disadvantage is the rigidity of the method. The Lowry method requires precisely-timed additions of reagent, immediate vortexing and prolonged incubation. Furthermore, the Lowry method also suffers from poor reproducibility and interference from a number of commonly used laboratory agents. Attempts to simplify the Lowry method have not, so far, been successful. Consequently, a need exists for a more flexible and rapid method for determination of protein.
In a recent modification, Smith et al (Anal. Biochem. 150, 76-85, 1985) combined the reaction of protein with alkaline copper with bicinchoninic acid. Although Smith et al's method has several advantages over the Lowry method, it suffers from lack of end-point in the reaction. The colour yield of the reaction continues to increase at a rate of 2-3% every ten minutes. Consequently, this method is not very accurate and the problem is compounded if a large number of samples are analysed in a single batch. In addition, Smith et al's method, using bicinchoninic acid, is a slow reaction requiring heating and a prolonged incubation period, which makes the method time-consuming.